Three longitudinal data points on CV parameters and plasma inflammatory markers have been collected. We are initiating the fourth measurement at the present time. In the cross sectional arm rats have been sacrificed at baseline, at 4 and 8 months of treatment, and again in November at 33 mo of age. Longitudinal Assessment of Cardiac Structure and Function is Derived from Repeated Echocardiograms: Based on 3 longitudinal measurements the following variables had significant interaction terms between Time and Drug (Placebo vs. ARA290) in respective random linear mixed effect models of ARA290 treatment over 3 time points: blood pressure, intraventricular septum-thickness, left ventricular internal diameter-systole, ejection fraction, and heart rate. Mitochondrial Fitness and mPTP-ROS Threshold: The mPTP ROS-threshold declines in cardiac mitochondria of aging rats and can serve as a biomarker of aging and mitochondrial fitness. One hour after a 10 min exposure to either EPO or ARA290, the mPTP ROS-threshold in cardiomyocytes (CM) from 3-month old rats was increased by 40-45% compared to time-matched controls, dependent on PI3K/AKT/GSK3 signaling. Further, the mPTP-ROS threshold response in CM to a short (10 min) exposure to ARA290 persists for several hours following ARA290 removal, conferring a biological-response memory in the improvement of mitochondrial fitness. We will quantify changes in mitochondrial fitness during aging in CM to test whether chronic ARA290 treatment prevents or slows that age-related decline in mPTP-ROS thresholds. Aortic Histological Analysis: Aortae at baseline and four months of treatment time were harvested and have been prepared for morphometric analyses and quantification of collagen, elastin and elastin fragmentation. Invasion and proliferation of smooth muscle cells in vitro will be measured. Serum analysis: All assays have been evaluated using early-time samples to validate their stringency prior to the collection of the later aged samples at which separation between treatment and placebo groups is expected. Proteomic analysis (RayBiotech) involves quantitative screening for 67 inflammatory factors, growth factors, chemokines, receptors, and cytokines. For the metabolite survey, samples were sent to UC Davis for untargeted analysis of primary metabolism. We plan to make use of metabolomics analysis for the entire serum collection at the conclusion of the study. Tissue Biomarkers of Oxidative Stress: After 4 months of treatment with vehicle or ARA290, protein carbonylation and 4-HNE levels were measured as biomarkers of oxidative stress. The results indicate a significant increase in protein carbonylation in the kidney but not liver of treated rats (p<0.001, n=10). Similar trend was observed for 4-HNE-modified proteins by Western blotting. Under these conditions, there was no significant change in the level of protein carbonylation in the serum of treated rats as compared to vehicle-treated controls. Molecular Effects in Immune Cells: Blood samples have been subjected to 9-Color Flow Cytometric analysis of almost all adaptive and immune cell type populations. To date, no significant differences were evident between the two groups when considering the different cell types and their population percentages. RNA was purified from PBMCs, smooth muscle cells, and aortic tissue. Because there is limited information on rat primers, we are still in the process of designing functional segments. Once the primers are created, the samples will be analyzed through qPCR and sequencing to see global differences in gene profiles between the two experimental groups. Over the next year we will complete the repeated measurements at various time points At the study termination we will perform comprehensive analyses of longitudinal and cross sectional in vivo and ex vivo data, and cross sectional analyses of data from kidney, liver, skeletalmuscle and molecular immune cells. We plan to apply for a renewal for this project in order to complete, over the next year, the aforementioned implementation of projects and final data analysis.